Human Beta Casein Fragment (54–59) Modulates <em>M. bovis</em> BCG Survival and Basic Transcription Factor 3 (BTF3) Expression in THP-1 Cell Line

<div><p>Immunostimulatory peptides potentiate the immune system of the host and are being used as a viable adjunct to established therapeutic modalities in treatment of cancer and microbial infections. Several peptides derived from milk protein have been reported to induce immunostimulatory activity. Human β -casein fragment (54–59), natural sequence peptide (NS) carrying the Val-Glu-Pro-Ile-Pro-Tyr amino acid residues, was reported to activate the macrophages and impart potent immunostimulatory activity. In present study, we found that this peptide increases the clearance of <em>M. bovis</em> BCG from THP-1 cell line in vitro. The key biomolecules, involved in the clearance of BCG from macrophage like, nitric oxide, pro-inflammatory cytokines and chemokines, were not found to be significantly altered after peptide treatment in comparison to the untreated control. Using proteomic approach we found that BTF3a, an isoform of the Basic Transcription Factor, BTF3, was down regulated in THP-1 cell line after peptide treatment. This was reconfirmed by real time RT-PCR and western blotting. We report the BTF3a as a novel target of this hexapeptide. Based on the earlier findings and the results from the present studies, we suggest that the down regulation of BTF3a following the peptide treatment may augment the <em>M. bovis</em> BCG mediated apoptosis resulting in enhanced clearance of <em>M. bovis</em> BCG from THP-1 cell line.</p> </div

BTF3a is down regulated after NS treatment.

<p>Real Time RT-PCR (a) and western blotting (b) was performed to analyse the expression of BTF3. BTF3a expression was down regulated after NS and LPS treatment. Fold change in expression during treated conditions is presented in respect to control. The values and error bars represent average and standard deviations of three independent set of experiments. Student T test was performed to find out significant difference between control and treated conditions and comparisons were considered significantly different at P<0.01 (**). In western, two different isoforms of BTF3 were detected as shown in figure (Lanes: a - control, b - NS-20 µM, c - LPS-1 µg/ml). Lower panel depicts the equal level of actin protein which was taken as loading control.</p

Clearance of <i>M. bovis</i> BCG from THP-1.

<p>PMA differentiated THP-1 cells were treated with NS at 10 µM and 20 µM and infected with bioluminescent recombinant BCG. RLU was measured at 0, 24 and 48 hrs time point of infection. The percent decrease in RLU reading after 24 hrs and 48 hrs of infection was considered as percent clearance of bacilli from THP-1 over this time period of infection. Percent increase in clearance in treated cells was calculated in reference to control cells. Lipopolysaccharide (LPS) was taken as positive control for the study. The values and error bars represent average and standard deviations of three independent set of experiments.</p

Nitric oxide production from THP-1 after <i>M. bovis</i> BCG infection.

<p>PMA differentiated THP-1 cells pretreated with NS at 10 µM and 20 µM were infected with recombinant BCG. Infection was continued in the presence of peptide for 24 and 48 hrs. Nitric oxide produced in cell culture after 24 and 48 hrs of infection was measured as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045905#s4" target="_blank">materials and methods</a>. Lipopolysaccharide (LPS) was taken as a positive control. The values and error bars represent average and standard deviations of three independent set of experiments. Student T test was performed to find out significant difference between control and treated group at P<0.01–0.05.</p

Change in pro-inflammatory cytokine and Chemokine expression from THP-1 upon peptide treatment.

<p>PMA differentiated THP-1 cells were treated with NS at 10 µM and 20 µM, Lipopolysaccharide (LPS) at 1 µg/ml concentration for 6 hrs. Change in TNF-α, IL-8, MIP-1beta, MCP-1 & Rantes expression level was quantified by real-time RT-PCR and expressed as fold change in expression of cytokines and cytokines in treated cells verses control cells. LPS was taken as a positive control. Bar shows the mean fold change ± SEM from three different experiments. The difference in cytokine expression between experimental group and control group was assessed by Student’s t-test and comparisons were considered significantly different at P≤0.05 (*) and at P<0.01 (**).</p